Proc. Nati. Acad. Sci. USA
Vol. 74, No. 12, pp. 5468-5-471, December 1977 Biochemistry
Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase (xanthine oxidase)
PHILIP T. PIENKOS, VINOD K. SHAH, AND WINSTON J. BRILL Department of Bacteriology and Center for Studies of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
Communicated by Robert H. Burris, October 3, 1977 ABSTRACT A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.62) in an extract from Neurospora crassa mutant strain Nit-i; however, FeMo-co is unable to activate nitrate reductase in strain Nit-i. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These'results indicate' that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase. A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N2 and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both'FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-i by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.
strains analogous to Nit-i. Acid-treated component I of nitrogenase activated inactive component I in extracts of mutant strain UW45 (11). Recently, Shah and Brill (13) isolated an iron-molybdenum cofactor (FeMo-co)* from nitrogenase component I. When our attempts to activate inactive nitrate reductase in extracts of N. crassa strain Nit-I by using FeMo-co failed, we reexamined the activation of nitrate reductase in extracts of strain Nit-1 by acid-treated component I. Contrary to the published data (4-7), we found evidence that acid-treated component I of nitrogenase cannot activate inactive nitrate reductase in extracts of mutant strain Nit-i.
MATERIALS AND METHODS The wild-type organism used was A. vinelandii OP. The mutant strain UW45, which does not fix N2, has been described (11). The conditions for growth, derepression of nitrogenase synthesis, preparation of extracts, isolation of FeMo-co, conditions for activation, and assays have been reported (11-14). Component I of nitrogenase from A. vnelandii was crystallized by the method of Shah and Brill (15). Molybdenum contents were determined with an atomic absorption spectrophotometer equipped with a graphite furnace. N. crassa wild-type strain FGSC 354 and mutant strain Nit-I were obtained from P. A. Ketchum. The organism was grown in medium containing ammonium chloride and transferred to medium containing sodium nitrate to induce synthesis of nitrate reductase (8). Hyphae were broken in 0.1 M sodium phosphate, pH 7.2/5 mM EDTA/1 mM phenylmethylsulfonyl fluoride/i mM NADPH/1% NaCl with a Potter-Elvehjem tissue homogenizer. The extracts were stored in serum vials under a helium atmosphere at -20°. Acid-treated molybdoenzymes were prepared as reported (4) but the reagents used were flushed with helium, and acid treatment was carried out under a helium atmosphere. Cell-free extracts (1.5-2 mg of protein) were incubated with varying amounts of acid-treated molybdoenzymes, FeMo-co, or molybdenum cofactor (Mo-co) for 30 min at room temperature unless otherwise specified. After this preincubation, nitrate reductase assays were performed with NADPH and FAD as electron donor and carrier (16). The nitrite formed was measured colorimetrically (17) at 560 nm with a Gilford spectrophotometer. Xanthine oxidase, NADPH, FAD, and phenylmethylsulfonyl fluoride were obtained from Sigma Chemical Co., Inc. (St. Louis, MO). Other chemicals used were of ultrapure or analytical grade available commercially. Doubly glass-distilled
Evidence for a common genetic determinant for different molybdoenzymes was presented by Pateman et al. (1) and Scazzocchio et al. (2). The first biochemical evidence for a cofactor common to different molybdoenzymes came from Nason's group (3, 4). Ketchum et al. (3) and Nason et al. (4) reported in vitro activation of inactive nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) from a mutant strain (Nit-i) of Neurospora crassa by acid-treated molybdoenzymes such as xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), aldehyde oxidase (aldehyde:oxygen oxidoreductase, EC 1.2.3.1), sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1), and component I (Mo-Fe protein) of nitrogenase. Various investigators (4-7) have used acidtreated component I of nitrogenase to activate nitrate reductase in extracts of strain Nit-1, and some (5-10) have asserted that the activating factor is a low molecular weight compound, possibly containing a polypeptide. Nagatani et al. (11) screened extracts of mutant strains of Azotobacter vinelandii (12), defective in component I of nitrogenase, for activation by acid-treated component I and found
Abbreviations: FeMo-co, iron-molybdenum cofactor; Mo-co, molybdenum cofactor; NMF, N-methylformamide.' * The original report of the iron-molybdenum cofactor used the abbreviation FeMoCo. This has been changed to FeMo-Co since we do not want to give the impression that cobalt is associated with the cofactors.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adverttsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5468
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Pienkos et al.
5469
Table 1. In vitro activation of inactive nitrogenase and nitrate reductase Reconstituted nitrogenase Reconstituted nitrate Source of addition* activity, nmol C2H4/15 min reductase activity, nmol N02-/15 min A. vinelandii component I (80) A. vinelandii component I (80), aerobic Xanthine oxidase (1.4) Xanthine oxidase (1.4), aerobic R. rubrum component 1 (49.2)
Partially purified component I (91.2) Extract of NH4+-grown A. vinelandii (59.5) Extract of N2-grown A. vinelandii (68.2) Extract of N-starved strain UW45 (60.8) Extract of wild type N. crassa (8.0) Mo-co from xanthine oxidase (1.4) Mo-co-FeMo-co mixture from partially purified component 1 (28.5)
Section A 331 71 0 0 121 Section B 285 0 49 0 0 Section C 0
37.4
90
5.3
0.0 0.0 31.7 8.1 0.0 10.2 1.7 2.4 3.0 3.9
* Preparations in sections A and B were acid-treated anaerobically as stated in Materials and Methods. These preparations were exposed to air for 30 min for aerobic samples. Preparations in section C were in N-methylformamide. The values in parentheses are pmol of molybdenum in the source added to the activation assay.
water and acid-cleaned glassware were used when necessary. The method used to isolate Mo-co from xanthine oxidase was essentially the same as that used for the isolation of FeMo-co (13), except that, after the initial acidification with citric acid (final concentration, 30 mM), disodium hydrogen phosphate was not added, so that the precipitated proteins could be centrifuged for further treatment to isolate Mo-co. One unit of FeMo-co or Mo-co is defined as the amount required to produce 1 nmol of ethylene (nitrite) per 15 min under the reconstitution conditions used. RESULTS AND DISCUSSION The FeMo-co from crystalline component I of nitrogenase failed to activate nitrate reductase in extracts of N. crassa mutant strain Nit-i. Upon exposure to air, the activating ability of FeMo-co to activate nitrogenase in extracts of A. vinelandii mutant strain UW45 was completely abolished (13). Activation of nitrate reductase in extracts of strain Nit-i is normally performed under aerobic conditions (4), and therefore we considered the possibility that air-oxidized FeMo-co could activate nitrate reductase in strain Nit-i. Our attempts to activate inactive nitrate reductase in extracts of strain Nit-i by using air-oxidized FeMo-co also were unsuccessful. We reexamined in vitro activation of nitrate reductase by acid-treated molybdoenzymes. Contrary to the published data (4-7), we observed that acid-treated component I of nitrogenase failed to activate nitrate reductase in extracts of strain Nit-i (Table 1, section A). Acid-treated component I activated nitrogenase in extracts of mutant strain UW45. When the acidtreated component I was exposed to air for 30 min, the ability to activate nitrogenase in extracts of strain UW45 decreased. This air-oxidized preparation of acid-treated component I also failed to activate nitrate reductase in extracts of strain Nit-i. Acid-treated component I from Rhodospirillum rubrum also failed to activate nitrate reductase in extracts of strain Nit-i but did activate nitrogenase in extracts of strain UW45. These results suggest that component I of nitrogenase does not contain the activating factor required to activate nitrate reductase in extracts of strain Nit-i. Acid-treated xanthine oxidase activated
nitrate reductase in strain Nit-i but failed to activate nitrogenase in extracts of strain UW45 (H. H. Nagatani, Ph.D. thesis, 1973, University of Wisconsin). To explain the discrepancy between our results and other published data (4-7), we used partially purified component I instead of pure component I. During separation of component I and component II from crude extracts on a DEAE-cellulose column, a fraction eluted with 0.25 M NaCl/0.025 M Tris-HCl, pH 7.4, was used as the source of partially purified component I. This partially purified component I fraction contains many other proteins in addition to component I when tested by analytical gel electrophoresis. Acid-treated preparations of this partially purified component I activated both nitrate reductase in strain Nit-i and nitrogenase in strain UW45 (Table 1, section B). These data suggest that the component I used by other investigators (4-7) contained a contaminant which, upon acidtreatment, could activate inactive nitrate reductase in strain Nit-i. The FeMo-co is specific for nitrogenase, whereas Mo-co is found in xanthine oxidase, nitrate reductase, sulfite oxidase (18), and presumably aldehyde oxidase. To distinguish between the activating factor for the nitrate reductase system and that of the nitrogenase system, we have named the former Mo-co and the latter FeMo-co. This terminology implies that Mo-co contains no iron. This is based on the fact that sulfite oxidase (19, 20) and nitrate reductase (21) contain no nonheme iron; however, xanthine oxidase (22) does contain nonheme iron, but we do not yet know if Mo-co from this enzyme has iron. Acid-treated crude extracts of wild-type A. vnelandii grown on medium containing excess NH4+ activated nitrate reductase in strain Nit-1 but not nitrogenase in strain UW45. Acid-treated extracts of A. vinelandii derepressed for nitrogenase synthesis, on the other hand, activated both nitrate reductase and nitrogenase (Table 1, section B). These results demonstrate that the Mo-co is synthesized even when A. -vnelandii is grown in a medium containing excess ammonium, whereas the FeMo-co is synthesized only under conditions that derepress nitrogenase synthesis. Acid-treated extracts of A. vinelandii mutant strain UW45 derepressed for nitrogenase synthesis activated nitrate reductase in strain Nit-i but not nitrogenase in strain UW45. These data plus the observation that strain UW45 can utilize
5470
Biochemistry: Pienkos et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
C4
x
x
40 ._
0, (A
o3 I0
0
I)
LL
F raction
FIG. 1. Elution profile of FeMo-co and Mo-co from a Sephadex G-100 column in NMF. The column was eluted with NMF at a flow rate of 12 ml/hr; approximately 3.5-ml fractions were collected anaerobically. 0-0, FeMo-co; 0 0, Mo-co from partially purified component I; *.... Mo-co from buttermilk xanthine oxidase. ----
,
nitrate as the sole N source indicate that mutant strain UW45 can synthesize Mo-co but not FeMo-co. If Mo-co is a precursor of FeMo-co, then the mutant strain UW45 has a lesion in a gene required for the processing of Mo-co to FeMo-co, and the gene products necessary for modification of Mo-co into FeMo-co are controlled by ammonia repression. We attempted to isolate Mo-co by using essentially the same method used for the isolation of FeMo-co (13). Data in Table 1, section C, demonstrate that Mo-co can be isolated from xanthine oxidase by the method used for isolation of FeMo-co from nitrogenase. When we used the partially purified component I mentioned above, both Mo-co and FeMo-co were eluted in N-methylformamide (NMF). NMF, in concentrations greater than 0.4%, is inhibitory either to the reconstitution of nitrate reductase or to the activity of nitrate reductase itself; therefore, the NMF concentration was kept below 0.4% during activation of nitrate reductase in extracts of strain Nit-i. In an attempt to separate Mo-co and FeMo-co, we applied the NMF supernatant solution (containing both Mo-co and FeMo-co) of partially purified component I to a Sephadex G-100 column in NMF (13). The results demonstrate that Mo-co and FeMo-co can easily be separated from the mixture containing both (Fig. 1). Similar results were obtained when the NMF-eluted material (containing both Mo-co and FeMo-co) of partially purified component I from Klebsiella pneumoniae was applied to this column. We also applied a mixture of FeMo-co from A. vtnelandii component I of nitrogenase and Mo-co from xanthine oxidase to this column and demonstrated
(Fig. 1) that they also can be separated. On the basis of the elution profile of Mo-co from different sources and activation of inactive nitrate reductase in strain Nit-i, it seems that Mo-co from various sources are similar. We determined molybdenum concentration in the fractions eluted from the Sephadex column and observed that it follows the activity profile. Molybdenum concentrations in the peak FeMo-co and Mo-co fractions from A. vlnelandii were 3.1 and 0.07 nmol/ml, respectively. The peak Mo-co fraction from xanthine oxidase contained 0.09 nmol of molybdenum per ml. To compare the properties of Mo-co with those of FeMo-co, we subjected Mo-co to various conditions (Table 2). The storage temperature had no effect on the activating ability of FeMo-co (in NMF) as long as it was kept anaerobic (13). The same was true for Mo-co prepared from xanthine oxidase. When the
Mo-co was diluted anaerobically in Tris-HCl buffer (pH 7.4) and citrate/phosphate buffer (pH 5.8), it was stable in aqueous media when stored at 00. Similar preparations stored at 250 for 3 hr lost 40% and 60% of activating ability at pH 5.8 and 7.4, respectively. The FeMo-co diluted anaerobically (13) in these buffers lost about 80% of its activating ability in 2 hr at pH 5.8 and in 4 hr at pH 7.4. From these data it seems that the Mo-co is somewhat more stable in an aqueous environment compared to the FeMo-co. At present, we do not have an explanation for the observed increase in Mo-co activity during its storage in aqueous medium or in NMF. Brief exposure to air completely abolished the activating ability of FeMo-co (13). The Mo-co from xanthine oxidase also was oxygen labile (Table 2), but it was not as sensitive as FeMo-co. After 15-min exposure to air at 00, Mo-co lost about 60% of its activity whereas FeMo-co lost 100% in 1 min under the same conditions (13). On 15-min exposure to air at 250, Mo-co lost about 95% of its activity. From these data it seems Table 2. Stability of Mo-co Conditions Activating ability* Section At 126 Anaerobic, 3 days at -200 118 Anaerobic, 2 days at 00 112 Anaerobic, 24 hr at 250 37 Aerobic, 15 min at 00 4 Aerobic, 15 min at 250 Section Bt 110 Anaerobic, 24 hr at 00 60 Anaerobic, 3 hr at 250 Section C§ 102 Anaerobic, 24 hr at 00 40 Anaerobic, 3 hr at 250 * Based on activation of inactive nitrate reductase in extracts of N. crassa strain Nit-i. Activating ability of Mo-co in NMF assayed at 0 time is considered to be 100%. Mo-co was prepared from xanthine oxidase. t Mo-co in NMF. Mo-co in citrate/phosphate buffer, pH 5.8 (ionic strength, 0.45 p) containing 1.2 mM dithionite. § Mo-co in 25 mM Tris.HCl, pH 7.4, containing 1.2 mM dithionite.
Biochemistry:
Pienkos et al.
that temperature has a pronounced effect on oxygen sensitivity of Mo-co. Mo-co and FeMo-co have similar properties: stability in NMF, sensitivity to 02, and instability in aqueous environments. But there are differences between these two cofactors; one very important difference is molecular weight as judged by Sephadex G-100 column chromatography. A most intriguing question remains to be answered: since Mo-co and FeMo-co appear to coexist in nitrogen-fixing organisms, what is the relationship between these two cofactors? Is Mo-co a direct precursor of FeMo-co? We sincerely appreciate the technical assistance of John Chisnell and the use of the atomic absorption spectrophotometer belonging to Dr. Frank Siegel. Nitrogenase component I from R. rubrum was a gift from Paul Ludden. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by grants from the National Institutes of Health (GM 22130) and the National Science Foundation (PCM 74-01134). P.T.P. was supported by National Institutes of Health Cellular and Molecular Biology Training Grant GM 07215. 1. Pateman, J. A., Cove, D. J., Rever, B. M. & Roberts, D. B. (1964) Nature 201,58-60. 2. Scazzocchio, G., Holl, F. G. & Foguelman, A. I. (1973) Eur. J. Biochem. 36, 428-445. 3. Ketchum, P. A., Cambier, H. Y., Frazier, W. A. III, Madansky, C. H. & Nason, A (1970) Proc. Natl. Acad. Sci. USA 66,10161023. 4. Nason, A., Lee, K. Y., Pan, S. S., Ketchum, P. A., Lamberti, A. & DeVries, J. (1971) Proc. Natl. Acad. Sci. USA 68, 32423246. 5. Lvov, N. P., Ganelin, V. L., Alikulov, Z. & Kretovich, V. L. (1975) J. Acad. Sci. USSR 3,371-376.
Proc. Natl. Acad. Sci. USA 74 (1977)
5471
6. McKenna, C., Lvov, N. P., Ganelin, V. L., Sergeev, N. S. & Kretovich, V. L. (1974) Dokl. Akad. Nauk. SSSR 217,228-231. 7. Zumft, W. G. (1974) Ber. Dtsch. Bot. Ges. 87, 135-143. 8. Ketchum, P. A. & Sevilla, C. L. (1973) J. Bacteriol. 116, 600609. 9. Ketchum, P. A. & Swarin, R. S. (1973) Biochem. Biophys. Res. Commun. 52, 1450-1456. 10. Ganelin, V. L., Lvov, N. P., Sergeev, N. S., Shaposhnikov, G. L. & Kretovich, V. L. (1972) Dokl. Akad. Nauk. SSSR 26, 12361238. 11. Nagatani, H. H., Shah, V. K. & Brill, W. J. (1974) J. Bacteriol.
120,697-701.
12. Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H. & Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255. 13. Shah, V. K. & Brill, W. J. (1977) Proc. Nat!. Acad. Sci. USA 74, 3249-3253. 14. Shah, V. K., Davis, L. C. & Brill, W. J. (1972) Biochim. Bwphys. Acta 256, 498-511. 15. Shah, V. K. & Brill, W. J. (1973) Biochim. Biophys. Acta 305, 445-454. 16. Garrett, R. H. & Nason, A. (1967) Proc. Nat!. Acad. Sci. USA 58, 1603-1610. 17. Nicholas, D. J. D. & Nason, A. (1957) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 3, p. 983. 18. Johnson, J. L., Jones, H. P. & Rajagopalan, K. V. (1977) J. Biol. Chem. 252, 4994-5003. 19. Cohen, H. J. & Fridovich, I. (1971) J. Biol. Chem. 246, 367373. 20. Cohen, H. J., Fridovich, I. & Rajagopalan, K. V. (1971) J. Biol.
Chem. 246, 374-382. 21. Garrett, R. H. & Nason, A. (1969) J. Biol. Chem. 244, 28702882. 22. Brady, F. O., Rajagopalan, K. V. & Handler, P. (1971) in Flavins and Flavoproteins, ed. Kamin, H. (University Park Press, Baltimore, MD), pp. 425-446.
Vol. 74, No. 12, pp. 5468-5-471, December 1977 Biochemistry
Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase (xanthine oxidase)
PHILIP T. PIENKOS, VINOD K. SHAH, AND WINSTON J. BRILL Department of Bacteriology and Center for Studies of Nitrogen Fixation, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
Communicated by Robert H. Burris, October 3, 1977 ABSTRACT A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.62) in an extract from Neurospora crassa mutant strain Nit-i; however, FeMo-co is unable to activate nitrate reductase in strain Nit-i. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These'results indicate' that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase. A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N2 and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both'FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-i by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.
strains analogous to Nit-i. Acid-treated component I of nitrogenase activated inactive component I in extracts of mutant strain UW45 (11). Recently, Shah and Brill (13) isolated an iron-molybdenum cofactor (FeMo-co)* from nitrogenase component I. When our attempts to activate inactive nitrate reductase in extracts of N. crassa strain Nit-I by using FeMo-co failed, we reexamined the activation of nitrate reductase in extracts of strain Nit-1 by acid-treated component I. Contrary to the published data (4-7), we found evidence that acid-treated component I of nitrogenase cannot activate inactive nitrate reductase in extracts of mutant strain Nit-i.
MATERIALS AND METHODS The wild-type organism used was A. vinelandii OP. The mutant strain UW45, which does not fix N2, has been described (11). The conditions for growth, derepression of nitrogenase synthesis, preparation of extracts, isolation of FeMo-co, conditions for activation, and assays have been reported (11-14). Component I of nitrogenase from A. vnelandii was crystallized by the method of Shah and Brill (15). Molybdenum contents were determined with an atomic absorption spectrophotometer equipped with a graphite furnace. N. crassa wild-type strain FGSC 354 and mutant strain Nit-I were obtained from P. A. Ketchum. The organism was grown in medium containing ammonium chloride and transferred to medium containing sodium nitrate to induce synthesis of nitrate reductase (8). Hyphae were broken in 0.1 M sodium phosphate, pH 7.2/5 mM EDTA/1 mM phenylmethylsulfonyl fluoride/i mM NADPH/1% NaCl with a Potter-Elvehjem tissue homogenizer. The extracts were stored in serum vials under a helium atmosphere at -20°. Acid-treated molybdoenzymes were prepared as reported (4) but the reagents used were flushed with helium, and acid treatment was carried out under a helium atmosphere. Cell-free extracts (1.5-2 mg of protein) were incubated with varying amounts of acid-treated molybdoenzymes, FeMo-co, or molybdenum cofactor (Mo-co) for 30 min at room temperature unless otherwise specified. After this preincubation, nitrate reductase assays were performed with NADPH and FAD as electron donor and carrier (16). The nitrite formed was measured colorimetrically (17) at 560 nm with a Gilford spectrophotometer. Xanthine oxidase, NADPH, FAD, and phenylmethylsulfonyl fluoride were obtained from Sigma Chemical Co., Inc. (St. Louis, MO). Other chemicals used were of ultrapure or analytical grade available commercially. Doubly glass-distilled
Evidence for a common genetic determinant for different molybdoenzymes was presented by Pateman et al. (1) and Scazzocchio et al. (2). The first biochemical evidence for a cofactor common to different molybdoenzymes came from Nason's group (3, 4). Ketchum et al. (3) and Nason et al. (4) reported in vitro activation of inactive nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) from a mutant strain (Nit-i) of Neurospora crassa by acid-treated molybdoenzymes such as xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), aldehyde oxidase (aldehyde:oxygen oxidoreductase, EC 1.2.3.1), sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1), and component I (Mo-Fe protein) of nitrogenase. Various investigators (4-7) have used acidtreated component I of nitrogenase to activate nitrate reductase in extracts of strain Nit-1, and some (5-10) have asserted that the activating factor is a low molecular weight compound, possibly containing a polypeptide. Nagatani et al. (11) screened extracts of mutant strains of Azotobacter vinelandii (12), defective in component I of nitrogenase, for activation by acid-treated component I and found
Abbreviations: FeMo-co, iron-molybdenum cofactor; Mo-co, molybdenum cofactor; NMF, N-methylformamide.' * The original report of the iron-molybdenum cofactor used the abbreviation FeMoCo. This has been changed to FeMo-Co since we do not want to give the impression that cobalt is associated with the cofactors.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adverttsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5468
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Pienkos et al.
5469
Table 1. In vitro activation of inactive nitrogenase and nitrate reductase Reconstituted nitrogenase Reconstituted nitrate Source of addition* activity, nmol C2H4/15 min reductase activity, nmol N02-/15 min A. vinelandii component I (80) A. vinelandii component I (80), aerobic Xanthine oxidase (1.4) Xanthine oxidase (1.4), aerobic R. rubrum component 1 (49.2)
Partially purified component I (91.2) Extract of NH4+-grown A. vinelandii (59.5) Extract of N2-grown A. vinelandii (68.2) Extract of N-starved strain UW45 (60.8) Extract of wild type N. crassa (8.0) Mo-co from xanthine oxidase (1.4) Mo-co-FeMo-co mixture from partially purified component 1 (28.5)
Section A 331 71 0 0 121 Section B 285 0 49 0 0 Section C 0
37.4
90
5.3
0.0 0.0 31.7 8.1 0.0 10.2 1.7 2.4 3.0 3.9
* Preparations in sections A and B were acid-treated anaerobically as stated in Materials and Methods. These preparations were exposed to air for 30 min for aerobic samples. Preparations in section C were in N-methylformamide. The values in parentheses are pmol of molybdenum in the source added to the activation assay.
water and acid-cleaned glassware were used when necessary. The method used to isolate Mo-co from xanthine oxidase was essentially the same as that used for the isolation of FeMo-co (13), except that, after the initial acidification with citric acid (final concentration, 30 mM), disodium hydrogen phosphate was not added, so that the precipitated proteins could be centrifuged for further treatment to isolate Mo-co. One unit of FeMo-co or Mo-co is defined as the amount required to produce 1 nmol of ethylene (nitrite) per 15 min under the reconstitution conditions used. RESULTS AND DISCUSSION The FeMo-co from crystalline component I of nitrogenase failed to activate nitrate reductase in extracts of N. crassa mutant strain Nit-i. Upon exposure to air, the activating ability of FeMo-co to activate nitrogenase in extracts of A. vinelandii mutant strain UW45 was completely abolished (13). Activation of nitrate reductase in extracts of strain Nit-i is normally performed under aerobic conditions (4), and therefore we considered the possibility that air-oxidized FeMo-co could activate nitrate reductase in strain Nit-i. Our attempts to activate inactive nitrate reductase in extracts of strain Nit-i by using air-oxidized FeMo-co also were unsuccessful. We reexamined in vitro activation of nitrate reductase by acid-treated molybdoenzymes. Contrary to the published data (4-7), we observed that acid-treated component I of nitrogenase failed to activate nitrate reductase in extracts of strain Nit-i (Table 1, section A). Acid-treated component I activated nitrogenase in extracts of mutant strain UW45. When the acidtreated component I was exposed to air for 30 min, the ability to activate nitrogenase in extracts of strain UW45 decreased. This air-oxidized preparation of acid-treated component I also failed to activate nitrate reductase in extracts of strain Nit-i. Acid-treated component I from Rhodospirillum rubrum also failed to activate nitrate reductase in extracts of strain Nit-i but did activate nitrogenase in extracts of strain UW45. These results suggest that component I of nitrogenase does not contain the activating factor required to activate nitrate reductase in extracts of strain Nit-i. Acid-treated xanthine oxidase activated
nitrate reductase in strain Nit-i but failed to activate nitrogenase in extracts of strain UW45 (H. H. Nagatani, Ph.D. thesis, 1973, University of Wisconsin). To explain the discrepancy between our results and other published data (4-7), we used partially purified component I instead of pure component I. During separation of component I and component II from crude extracts on a DEAE-cellulose column, a fraction eluted with 0.25 M NaCl/0.025 M Tris-HCl, pH 7.4, was used as the source of partially purified component I. This partially purified component I fraction contains many other proteins in addition to component I when tested by analytical gel electrophoresis. Acid-treated preparations of this partially purified component I activated both nitrate reductase in strain Nit-i and nitrogenase in strain UW45 (Table 1, section B). These data suggest that the component I used by other investigators (4-7) contained a contaminant which, upon acidtreatment, could activate inactive nitrate reductase in strain Nit-i. The FeMo-co is specific for nitrogenase, whereas Mo-co is found in xanthine oxidase, nitrate reductase, sulfite oxidase (18), and presumably aldehyde oxidase. To distinguish between the activating factor for the nitrate reductase system and that of the nitrogenase system, we have named the former Mo-co and the latter FeMo-co. This terminology implies that Mo-co contains no iron. This is based on the fact that sulfite oxidase (19, 20) and nitrate reductase (21) contain no nonheme iron; however, xanthine oxidase (22) does contain nonheme iron, but we do not yet know if Mo-co from this enzyme has iron. Acid-treated crude extracts of wild-type A. vnelandii grown on medium containing excess NH4+ activated nitrate reductase in strain Nit-1 but not nitrogenase in strain UW45. Acid-treated extracts of A. vinelandii derepressed for nitrogenase synthesis, on the other hand, activated both nitrate reductase and nitrogenase (Table 1, section B). These results demonstrate that the Mo-co is synthesized even when A. -vnelandii is grown in a medium containing excess ammonium, whereas the FeMo-co is synthesized only under conditions that derepress nitrogenase synthesis. Acid-treated extracts of A. vinelandii mutant strain UW45 derepressed for nitrogenase synthesis activated nitrate reductase in strain Nit-i but not nitrogenase in strain UW45. These data plus the observation that strain UW45 can utilize
5470
Biochemistry: Pienkos et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
C4
x
x
40 ._
0, (A
o3 I0
0
I)
LL
F raction
FIG. 1. Elution profile of FeMo-co and Mo-co from a Sephadex G-100 column in NMF. The column was eluted with NMF at a flow rate of 12 ml/hr; approximately 3.5-ml fractions were collected anaerobically. 0-0, FeMo-co; 0 0, Mo-co from partially purified component I; *.... Mo-co from buttermilk xanthine oxidase. ----
,
nitrate as the sole N source indicate that mutant strain UW45 can synthesize Mo-co but not FeMo-co. If Mo-co is a precursor of FeMo-co, then the mutant strain UW45 has a lesion in a gene required for the processing of Mo-co to FeMo-co, and the gene products necessary for modification of Mo-co into FeMo-co are controlled by ammonia repression. We attempted to isolate Mo-co by using essentially the same method used for the isolation of FeMo-co (13). Data in Table 1, section C, demonstrate that Mo-co can be isolated from xanthine oxidase by the method used for isolation of FeMo-co from nitrogenase. When we used the partially purified component I mentioned above, both Mo-co and FeMo-co were eluted in N-methylformamide (NMF). NMF, in concentrations greater than 0.4%, is inhibitory either to the reconstitution of nitrate reductase or to the activity of nitrate reductase itself; therefore, the NMF concentration was kept below 0.4% during activation of nitrate reductase in extracts of strain Nit-i. In an attempt to separate Mo-co and FeMo-co, we applied the NMF supernatant solution (containing both Mo-co and FeMo-co) of partially purified component I to a Sephadex G-100 column in NMF (13). The results demonstrate that Mo-co and FeMo-co can easily be separated from the mixture containing both (Fig. 1). Similar results were obtained when the NMF-eluted material (containing both Mo-co and FeMo-co) of partially purified component I from Klebsiella pneumoniae was applied to this column. We also applied a mixture of FeMo-co from A. vtnelandii component I of nitrogenase and Mo-co from xanthine oxidase to this column and demonstrated
(Fig. 1) that they also can be separated. On the basis of the elution profile of Mo-co from different sources and activation of inactive nitrate reductase in strain Nit-i, it seems that Mo-co from various sources are similar. We determined molybdenum concentration in the fractions eluted from the Sephadex column and observed that it follows the activity profile. Molybdenum concentrations in the peak FeMo-co and Mo-co fractions from A. vlnelandii were 3.1 and 0.07 nmol/ml, respectively. The peak Mo-co fraction from xanthine oxidase contained 0.09 nmol of molybdenum per ml. To compare the properties of Mo-co with those of FeMo-co, we subjected Mo-co to various conditions (Table 2). The storage temperature had no effect on the activating ability of FeMo-co (in NMF) as long as it was kept anaerobic (13). The same was true for Mo-co prepared from xanthine oxidase. When the
Mo-co was diluted anaerobically in Tris-HCl buffer (pH 7.4) and citrate/phosphate buffer (pH 5.8), it was stable in aqueous media when stored at 00. Similar preparations stored at 250 for 3 hr lost 40% and 60% of activating ability at pH 5.8 and 7.4, respectively. The FeMo-co diluted anaerobically (13) in these buffers lost about 80% of its activating ability in 2 hr at pH 5.8 and in 4 hr at pH 7.4. From these data it seems that the Mo-co is somewhat more stable in an aqueous environment compared to the FeMo-co. At present, we do not have an explanation for the observed increase in Mo-co activity during its storage in aqueous medium or in NMF. Brief exposure to air completely abolished the activating ability of FeMo-co (13). The Mo-co from xanthine oxidase also was oxygen labile (Table 2), but it was not as sensitive as FeMo-co. After 15-min exposure to air at 00, Mo-co lost about 60% of its activity whereas FeMo-co lost 100% in 1 min under the same conditions (13). On 15-min exposure to air at 250, Mo-co lost about 95% of its activity. From these data it seems Table 2. Stability of Mo-co Conditions Activating ability* Section At 126 Anaerobic, 3 days at -200 118 Anaerobic, 2 days at 00 112 Anaerobic, 24 hr at 250 37 Aerobic, 15 min at 00 4 Aerobic, 15 min at 250 Section Bt 110 Anaerobic, 24 hr at 00 60 Anaerobic, 3 hr at 250 Section C§ 102 Anaerobic, 24 hr at 00 40 Anaerobic, 3 hr at 250 * Based on activation of inactive nitrate reductase in extracts of N. crassa strain Nit-i. Activating ability of Mo-co in NMF assayed at 0 time is considered to be 100%. Mo-co was prepared from xanthine oxidase. t Mo-co in NMF. Mo-co in citrate/phosphate buffer, pH 5.8 (ionic strength, 0.45 p) containing 1.2 mM dithionite. § Mo-co in 25 mM Tris.HCl, pH 7.4, containing 1.2 mM dithionite.
Biochemistry:
Pienkos et al.
that temperature has a pronounced effect on oxygen sensitivity of Mo-co. Mo-co and FeMo-co have similar properties: stability in NMF, sensitivity to 02, and instability in aqueous environments. But there are differences between these two cofactors; one very important difference is molecular weight as judged by Sephadex G-100 column chromatography. A most intriguing question remains to be answered: since Mo-co and FeMo-co appear to coexist in nitrogen-fixing organisms, what is the relationship between these two cofactors? Is Mo-co a direct precursor of FeMo-co? We sincerely appreciate the technical assistance of John Chisnell and the use of the atomic absorption spectrophotometer belonging to Dr. Frank Siegel. Nitrogenase component I from R. rubrum was a gift from Paul Ludden. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by grants from the National Institutes of Health (GM 22130) and the National Science Foundation (PCM 74-01134). P.T.P. was supported by National Institutes of Health Cellular and Molecular Biology Training Grant GM 07215. 1. Pateman, J. A., Cove, D. J., Rever, B. M. & Roberts, D. B. (1964) Nature 201,58-60. 2. Scazzocchio, G., Holl, F. G. & Foguelman, A. I. (1973) Eur. J. Biochem. 36, 428-445. 3. Ketchum, P. A., Cambier, H. Y., Frazier, W. A. III, Madansky, C. H. & Nason, A (1970) Proc. Natl. Acad. Sci. USA 66,10161023. 4. Nason, A., Lee, K. Y., Pan, S. S., Ketchum, P. A., Lamberti, A. & DeVries, J. (1971) Proc. Natl. Acad. Sci. USA 68, 32423246. 5. Lvov, N. P., Ganelin, V. L., Alikulov, Z. & Kretovich, V. L. (1975) J. Acad. Sci. USSR 3,371-376.
Proc. Natl. Acad. Sci. USA 74 (1977)
5471
6. McKenna, C., Lvov, N. P., Ganelin, V. L., Sergeev, N. S. & Kretovich, V. L. (1974) Dokl. Akad. Nauk. SSSR 217,228-231. 7. Zumft, W. G. (1974) Ber. Dtsch. Bot. Ges. 87, 135-143. 8. Ketchum, P. A. & Sevilla, C. L. (1973) J. Bacteriol. 116, 600609. 9. Ketchum, P. A. & Swarin, R. S. (1973) Biochem. Biophys. Res. Commun. 52, 1450-1456. 10. Ganelin, V. L., Lvov, N. P., Sergeev, N. S., Shaposhnikov, G. L. & Kretovich, V. L. (1972) Dokl. Akad. Nauk. SSSR 26, 12361238. 11. Nagatani, H. H., Shah, V. K. & Brill, W. J. (1974) J. Bacteriol.
120,697-701.
12. Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H. & Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255. 13. Shah, V. K. & Brill, W. J. (1977) Proc. Nat!. Acad. Sci. USA 74, 3249-3253. 14. Shah, V. K., Davis, L. C. & Brill, W. J. (1972) Biochim. Bwphys. Acta 256, 498-511. 15. Shah, V. K. & Brill, W. J. (1973) Biochim. Biophys. Acta 305, 445-454. 16. Garrett, R. H. & Nason, A. (1967) Proc. Nat!. Acad. Sci. USA 58, 1603-1610. 17. Nicholas, D. J. D. & Nason, A. (1957) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 3, p. 983. 18. Johnson, J. L., Jones, H. P. & Rajagopalan, K. V. (1977) J. Biol. Chem. 252, 4994-5003. 19. Cohen, H. J. & Fridovich, I. (1971) J. Biol. Chem. 246, 367373. 20. Cohen, H. J., Fridovich, I. & Rajagopalan, K. V. (1971) J. Biol.
Chem. 246, 374-382. 21. Garrett, R. H. & Nason, A. (1969) J. Biol. Chem. 244, 28702882. 22. Brady, F. O., Rajagopalan, K. V. & Handler, P. (1971) in Flavins and Flavoproteins, ed. Kamin, H. (University Park Press, Baltimore, MD), pp. 425-446.
